Clostridia are gram positive, anaerobic and low GC bacteria that are widely used in industry for their capacities to produce solvents, in particular butanol, ethanol and acetone, but also diols like 1,3 propanediol, organic acids like acetic, butyric or lactic acid and vaccines.
Construction of recombinant Clostridia is an important part of the development in the field. Clostridium strains are genetically modified in order to improve their industrial capabilities.
To perform these modifications, homologous recombination is the most used technique in all kinds of organisms. Transformation and homologous recombination in several microorganisms have been extensively described in the art. See for example (Datsenko and Wanner; PNAS, 2000) and (Fabret et al., Molecular Microbiology, 2002).
Clostridia are not naturally transformable and currently available methods for their transformation are inefficient and do not permit the introduction of multiple mutations. This has hampered industrial developments in this field.
Clostridia commonly produce extracellular DNAses and restriction enzymes that degrade foreign DNA before and after introduction into the cells for transformation. Classic methods based on the introduction of PCR fragments that work well in many microorganisms such as E. coli or yeast, are not feasible in these organisms, since the extracellular and intracellular half life of the DNA construct to be recombined is too short and recombination efficiency is generally low. In other organisms these difficulties have been circumvented by using vectors that replicate in the host thus increasing the likelihood of the recombination event. Nevertheless after the recombination event the vector that now carries the intact target DNA sequence has to be eliminated. This problem was solved in Lactococcus lactis (Biswas et al, J. Bacteriol., 1993) by using temperature-sensitive replicons that can be eliminated at the non-permissive temperature. No vectors with these characteristics are currently available for Clostridia. Therefore construction of mutants in Clostridia has so far been very laborious and often unsuccessful.
Inactivation of genes in Clostridia were reported in the following articles (see table 1).
TABLE 1StrainGenotypeReferenceClostridium acetobutylicumbuk-, MLSRGreen et al., 1996PJC4BKClostridium acetobutylicumpta-, MLSRGreen et al., 1996PJC4PTAClostridium acetobutylicumaad-, MLSRGreen andPJC4AADBennett, 1996Clostridium perfringensΔ cpe, CatPSarker et al., 1999SM101 and F4969Clostridium perfringens Strain 13Δ luxSOhtani et al., 2002Clostridium acetobutylicum SKO1ΔspoA, MLSRHarris et al., 2002Clostridium perfringens Type AΔ spo0AHuang et al., 2004Clostridium perfringens SM101ccpA-, CatPVarga et al., 2004Clostridium acetobutylicumΔSpoIIE,WO 2006/007530ATCC 824 buk-buk-, CatP
Gene inactivation was so far performed in Clostridia by transforming with circular DNA that could not replicate in the target strains. Since DNAses and DNA restriction endonucleases present in Clostridia rapidly degrade the introduced DNA, and generally the recombination frequency in this genus is not very high, the obtention of mutants has been very laborious.
In addition, the so far described recombinant strains (see above) are all resistant to MLS or chloramphenicol and the corresponding marker genes can not be removed after the recombination event has occurred. This limits the number of possible recombinations to the number of available resistance markers in these bacteria to a maximum of 3. Furthermore, for the industrial use of these bacteria, it might be useful to have markerless strains in order to avoid the release of antibiotic resistance genes into fermentation media.
Moreover, some of these strains that have been obtained by single recombination events have the disadvantage that they are not stable if cultured without any selection pressure.
Consequently, there is still a need in the state of the art for a method for the transformation of Clostridia with high efficiency, with an easy step of selection of recombinant strains, that allows successive DNA sequence replacements in the same strain, leading to recombinant Clostridia that are genetically stable and markerless.
The present invention is related to a new method for replacing or deleting DNA sequences in Clostridia, easy to perform and applicable at an industrial level. This method is useful to modify several genetic loci in Clostridia in a routine manner.
This method is based on a replicative vector useful for the transformation of Clostridia with high efficiency.
An unlimited number of mutations can be introduced into the genome with this new method, by eliminating resistance cassettes from the genome and reusing them in successive rounds of DNA sequence replacement.
Efficient introduction of multiple mutations into Clostridia should enable industry to improve existing industrial strains and to develop new processes.